Dynamic Membrane association of the bacterial SRP receptor in E. coli
Final Report Abstract
The recognition and transport of proteins destined to reside in different cellular compartments is an inherent and essential process in eukaryotic and prokaryotic cells. Although a large number of different protein transport pathways have been identified in different species, only a few pathways appear to be universally conserved. An example is the Signal recognition particle (SRP) dependent protein targeting pathway, which consists of SRP and the SRP-receptor (SR). SR in eukaryotes consists of two subunits; SRα binds SRP, while SRβ is an integral membrane protein that tethers SRα to the endoplasmic reticulum membrane. The bacterial SR consists only of the SRα homologue FtsY, but still significant amounts of FtsY were found associated with the bacterial cytoplasmic membrane, although a membrane-integral SRβ subunit is missing and FtsY itself does not contains a transmembrane domain. We have characterized the membrane binding of FtsY by multiple in vivo and in vitro approaches and found that FtsY binds to the membrane via two short lipid-binding helices with a preference for negatively charged lipids. In addition, FtsY binds to the SecY subunit of the universally conserved SecYEG translocon. Here, FtsY occupies the ribosome-binding site of the SecYEG translocon, which suggested a possible mechanism for hand-over of nascent membrane proteins from the targeting machinery to the insertion machinery. This was addressed by in site-directed cross-linking and in vitro FRET analyses, which revealed that SRP delivers RNCs to the SecY-bound FtsY, resulting in a quaternary complex, in which FtsY was displaced from the ribosome-binding SecY loop, which in turn was then occupied by the ribosomal protein uL23. Thus, the interaction between SRP and SR induces a reciprocal conformational change that exposes the ribosome-binding site on the SecYEG translocon and simultaneously the SecY binding site on the ribosome. This explains how a nascent membrane protein is handed over from the targeting machinery to the SecY translocon. SRP not delivers substrate to SecYEG, but also to the YidC insertase. YidC inserts not only small membrane proteins, as previously suggested, but also multispanning membrane proteins that were previously thought to be exclusively SecYEG dependent. Targeting of multi-spanning membrane proteins to YidC is also SRP-dependent, but appears to be not strictly FtsY-dependent. This suggests that SRP-dependent targeting to YidC does not exactly follow the pathway revealed for targeting to SecYEG.
Publications
- (2009) Predominant membrane localization is an essential feature of the bacterial SRP receptor. BMC Biology, 7, 76
Mircheva, M., Boy, D., Weiche, B., Hucke, F., Graumann, P., und Koch, H.G.
(See online at https://doi.org/10.1186/1741-7007-7-76) - (2009) The depletion of the SRP receptor inactivates ribosomes in Escherichia coli. J. Bact. 191, 7017-7026
Bürk, J., Weiche, B., Wenk, M., Boy, D., Nestel, S., Heimrich, B., und Koch, H.G.
(See online at https://doi.org/10.1128/JB.00208-09) - (2009) Two cooperating helices constitute the lipid binding domain of the bacterial SRP receptor. J. Mol. Biol. 390, 401-413
Braig, D., Bär C., Thumfart, J.O., und Koch, H.G.
(See online at https://doi.org/10.1016/j.jmb.2009.04.061) - (2009). Visualization of distinct entities of the SecYEG translocon during translocation and integration of bacterial proteins. Mol. Biol. Cell 20, 1804-1815
Boy, D. und Koch, H.G.
(See online at https://doi.org/10.1091/mbc.e08-08-0886) - (2011). The bacterial SRP receptor, SecA and the ribosome use overlapping binding sites on the SecY translocon. Traffic 12, 563-578
Kuhn, P., Weiche, B., Sturm, L., Sommer, E., Drepper, F.,Warscheid, B., Sourjik, V., und Koch, H.G.
(See online at https://doi.org/10.1111/j.1600-0854.2011.01167.x) - (2012). Promiscuous targeting of polytopic membrane proteins to SecYEG or YidC by the E. coli Signal recognition particle. Mol. Biol. Cell 23. 464-479
Welte, T., Kudva, R., Kuhn, P., Sturm, L., Braig, D., Müller, M., Warscheid, B., Drepper, F., and Koch, H.G.
(See online at https://doi.org/10.1091/mbc.e11-07-0590) - (2013). Protein translocation across the inner membrane of Gram-negative bacteria: The Sec and Tat dependent protein transport pathways. Research in Microbiology 164; 505-534 Special Issue on Bacterial protein secretion systems (C. Wandersman, ed.)
Kudva, R., Denks, K., Kuhn, P., Vogt,, A., Müller, M and Koch, H.G.
(See online at https://doi.org/10.1016/j.resmic.2013.03.016) - (2013). YidC occupies the lateral gate of the SecYEG translocon and is sequentially displaced by a nascent membrane protein. J. Biol. Chem. 288, 16295-16307
Sachelaru, I., Petriman, N.A., Kudva, R., Kuhn, P., Welte, T., Knapp, B., Drepper, F., Warscheid, B., and Koch, H.G.
(See online at https://doi.org/10.1074/jbc.M112.446583) - (2014) Dynamic interaction of the Sec translocon with the periplasmic chaperone PpiD. J. Biol. Chem. 289, 21706-21715
Sachelaru, I., Petriman, N.A., Kudva, R. and Koch, H.G.
(See online at https://doi.org/10.1074/jbc.M114.577916) - (2014). Targeting and integration of bacterial membrane proteins. In: Bacterial Membranes: Structural and Molecular Biology (Remaut, H., and Fronzes, R. eds). Horizon Press, Norwich, U.K.
Kuhn, P., Kudva, R., Welte, T., Sturm, L., and Koch, H.G.
- (2014). The Sec translocon mediated protein transport in prokaryotes and eukaryotes. Mol. Membrane Biology, 31, 58-84
Denks, K., Vogt, A.; Sachelaru, I., Petriman, N.A. Kudva, R. and Koch, H.G.
(See online at https://doi.org/10.3109/09687688.2014.907455) - (2011). Signal-sequence independent SRP-SR complex formation at the membrane suggests an alternative targeting pathway within the SRP cycle. Mol. Biol. Cell 22, 2309-2323
Braig, D., Mircheva, M., Sachelaru, I., van der Sluis, E.O., Sturm, L., Beckmann, R., und Koch, H.G.
(See online at https://doi.org/10.1091/mbc.e11-02-0152) - (2015) Repositioning of the signal recognition particle receptor on the translocon upon ribosome binding. J. Cell Biol.
Kuhn, P., Draycheva, A., Vogt, A., Petriman, N.A., Sturm, L., Drepper, F., Warscheid, B., Wintermeyer, W., and Koch, H.G.
(See online at https://doi.org/10.1083/jcb.201502103)
